The goal is to investigate the molecular basis of pancreas-specific and parotid-specific gene expression. During the previous grant period, human and mouse amylase genes were cloned and characterized. An amylase minigene which is expressed specifically in pancreas of transgenic mice was constructed. Modifications of this minigene will be used to identify sequences required for transcription in pancreas and parotid. The sequences responsible for response to insulin will also be identified. The tissue-specific expression of ribonuclease phospholipase A2 in parotid and pancreas will be compared with amylase. Trans-acting factors which bind to the tissue-specific regulatory sequences will be purified from nuclear extracts, using an exonuclease protection assay to monitor purification. We will also use a genetic approach to identify tissue-specific regulators, by selection of defective cell lines which lack individual factors. Complementation analysis of these mutants will provide an estimate of the number of factors required for transcription of the pancreatic amylase promoter. The mutant cell lines will be transfected with genomic DNA in order to isolate the genes encoding the tissue-specific regulatory factors. These investigations will contribute to our understanding of the molecular basis of tissue-specific and hormonal regulation of mammalian gene expression.